The retroviral polyprotein Gag is the essential factor in a large number of viral pathogens, such as human immunodeficiency virus type 1 (HIV-1) and oncoviruses, that promotes the budding of progeny viral shells after binding to the host plasma membrane, leading to the formation of immature daughter particles which mature further into infectious viruses. A multitude of molecular interactions involved in membrane budding result in a complexity of the ensuing molecular reorganizations that impedes our understanding of these processes. This lack in understanding in turn prevents the targeting of this important step in the reproduction of virus particles with therapeutic interventions. Here, we focus on two hypotheses. (I) Gag binding to the lipid bilayer results from a hierarchical sequence of molecular interactions between the protein and the plasma membrane: Electrostatic steering of the membrane-binding domain, MA, to the bilayer surface; lipid-specific interaction of MA with the phosphatidylinositoldiphosphate PI(4,5)P2; and hydrophobic membrane insertion of a myristoyl anchor on MA's N-terminus. (II) In the full-length Gag polyprotein of HIV-1, the flexibility of the linker regions between distinct Gag domains is key for controlling the molecular reorganization that leads to membrane budding. We assembled a cross-disciplinary team of investigators who combine cutting-edge biophysical characterization capabilities of the structural and dynamic properties of proteins at membrane interfaces, located in Dr. Lashes' group at Carnegie Mellon University (CMU), with expertise in Gag protein expression, manipulation and characterization represented by Dr. Rein's group at the National Cancer Institute (NCI) which also has advanced capabilities in characterizing subcellular localization of protein constructs and the ultra- structural morphology of assembly products. These groups will work closely together to determine, in Aim 1, the factors that control the association of MA with membranes in binding studies, structural characterization with neutron scattering and quantitative measurements of the dynamics of protein association with lipid bilayers with correlation spectroscopy. With these tools, we also aim at understanding the mechanisms that lead to the recruitment of specific lipids into the viral shell. Under Aim 2, we characterize the origin and the implications of the conformational reorganization of full-length Gag at membrane surfaces in experimental and computational studies. Our team of investigators with its complementing expertise is uniquely qualified to boost our understanding of the molecular processes involved in viral shell formation. The public health relevance of this work lies in the development of a solid understanding of viral envelope formation in the replication step of HIV-1 virus and other retroviral pathogens. Its broader impact is to provide new techniques for the structural characterization of membrane proteins associated with lipid bilayers in their physiologically relevant, thermally disordered state. PUBLIC HEALTH RELEVANCE: The multiplication of viruses such as HIV in infected cells depends on the association of a viral protein, Gag, with the cell's plasma membrane which is consumed to form new viral shells in a complex sequence of molecular reorganizations. In this research project, we will determine the overall process that leads to the formation of viral shells because a thorough understanding of this process will be valuable for the development of new therapeutic strategies for interfering with virus formation in infected cells.